洛蒙德链霉菌S015中cutR/cutS双组分调控系统对洛蒙真菌素合成的调控
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摘要
【背景】cutR/cutS双组分调控系统在链霉菌次级代谢过程中起重要作用。【目的】通过同源重组的方法在野生型S015菌株中分别敲除cutR和cutS,构建单基因缺失突变株,研究cutR/cutS双组分调控系统对洛蒙真菌素合成的调控。【方法】对突变株及野生型菌株的发酵产物进行高效液相色谱法(Highperformanceliquidchromatography,HPLC)分析,通过qPCR测定基因表达量的变化。【结果】HPLC产物分析发现,S015Δcut R和S015Δcut S中洛蒙真菌素产量分别达到了128.1±26.4 mg/L和61.8±4.5 mg/L,分别为野生型S015产量的11.5倍和5.5倍。qPCR检测发现,S015ΔcutR突变株中lomo14、lomo10、lphzB、lphzC、lphzE和lphzG的表达量分别达到野生型的1 151.7±88.8、110.5±5.8、129.3±7.7、380.2±34.6、348.2±42.1和299.8±38.2倍;S015ΔcutS突变株中lomo14、lomo10、lphzB、lphzC、lphzE和lphzG的表达量分别达到野生型的4.3±0.5、2.2±0.2、9.3±0.9、10.3±0.6、20.7±1.5和20.4±0.8倍。【结论】cutR/cutS双组分调控系统在洛蒙德链霉菌的洛蒙真菌素合成过程中对其合成途径核心基因和侧链修饰基因的表达有抑制作用,从而抑制其合成。
[Background] The cutR/cutS two component system plays a significant role in secondarymetabolite in Streptomyces. [Objective] The aim of the study is to investigate the function of the cutR/cutStwo component system in the production of lomofungin in Streptomyces lomondensis S015. [Methods]HPLC was used to analysis fermentation production, meanwhile accessing quantitative real-time PCR tomonitor the levels of gene expression. [Results] HPLC results indicated that the yield of lomofungin inS015ΔcutR and S015ΔcutS reach the total amount of 128.1±26.4 mg/L and 61.8±4.5 mg/L respectively,that is 11.5 and 5.5 times of the yield of wild type. Results of qPCR indicated that in S015ΔcutR mutantthe relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG reached respectively1 151.7±88.8, 110.5±5.8, 129.3±7.7, 380.2±34.6, 348.2±42.1 and 299.8±38.2 times of the wild type, and inS015ΔcutS mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG wererespectively 4.3±0.5, 2.2±0.2, 9.3±0.9, 10.3±0.6, 20.7±1.5 and 20.4±0.8 times. [Conclusion] The studyshows that the cutR and cutS negatively regulate several main synthetic genes and side-chain modificationgenes of lomofungin production in S. lomondensis, thereby reducing the production.
[Background] The cutR/cutS two component system plays a significant role in secondarymetabolite in Streptomyces. [Objective] The aim of the study is to investigate the function of the cutR/cutStwo component system in the production of lomofungin in Streptomyces lomondensis S015. [Methods]HPLC was used to analysis fermentation production, meanwhile accessing quantitative real-time PCR tomonitor the levels of gene expression. [Results] HPLC results indicated that the yield of lomofungin inS015ΔcutR and S015ΔcutS reach the total amount of 128.1±26.4 mg/L and 61.8±4.5 mg/L respectively,that is 11.5 and 5.5 times of the yield of wild type. Results of qPCR indicated that in S015ΔcutR mutantthe relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG reached respectively1 151.7±88.8, 110.5±5.8, 129.3±7.7, 380.2±34.6, 348.2±42.1 and 299.8±38.2 times of the wild type, and inS015ΔcutS mutant the relative gene expression of lomo14, lomo10, lphzB, lphzC, lphzE and lphzG wererespectively 4.3±0.5, 2.2±0.2, 9.3±0.9, 10.3±0.6, 20.7±1.5 and 20.4±0.8 times. [Conclusion] The studyshows that the cutR and cutS negatively regulate several main synthetic genes and side-chain modificationgenes of lomofungin production in S. lomondensis, thereby reducing the production.
引文
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[17]Sheng CL,Wang W,Hu HB,et al.Function of a methyltransferase gene lomo3 involved in the biosynthesis of lomofungin[J].Microbiology China,2016,43(3):575-582(in Chinese)盛超兰,王威,胡洪波,等.一个甲基转移酶基因lomo3在洛蒙真菌素生物合成途径中的功能[J].微生物学通报,2016,43(3):575-582
[18]Li S,Ye QM,Wang W,et al.High-performance liquid chromatography analysis of lomofungin in Streptomyces lomondensis[J].Journal of Liquid Chromatography&Related Technologies,2013,36(15):2059-2068
[19]Xie K,Peng HS,Hu HB,et al.OxyR,an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP72[J].Research in Microbiology,2013,168(10):646-653
[2]Laursen JB,Nielsen J.Phenazine natural products:biosynthesis,synthetic analogues,and biological activity[J].Chemical Reviews,2004,104(3):1663-1686
[3]Kim CG,Yu TW,Fryhle CB,et al.3-Amino-5-hydroxybenzoic acid synthase,the terminal enzyme in the formation of the precursor of m C7N units in rifamycin and related antibiotics[J].The Journal of Biological Chemistry,1998,273(11):6030-6040
[4]Qu L,Liu HZ,Hu HB,et al.The biosynthesis of natural phenazine[J].Chinese Journal of Antibiotics,2010,35(3):168-174(in Chinese)屈丽,刘宏志,胡洪波,等.天然吩嗪化合物的生物合成[J].中国抗生素杂志,2010,35(3):168-174
[5]Zhang CX,Sheng CL,Wang W,et al.Identification of the lomofungin biosynthesis gene cluster and associated flavin-dependent monooxygenase gene in Streptomyces lomondensis S015[J].PLoS One,2015,10(8):e136228
[6]Parsons JF,Greenhagen BT,Shi K,et al.Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa[J].Biochemistry,2007,46(7):1821-1828
[7]Gibson J,Sood A,Hogan DA.Pseudomonas aeruginosa-Candida albicans interactions:localization and fungal toxicity of a phenazine derivative[J].Applied and Environmental Microbiology,2009,75(2):504-513
[8]Mentel M,Ahuja EG,Mavrodi DV,et al.Of two make one:the biosynthesis of phenazines[J].Chembiochem,2009,10(14):2295-2304
[9]Mavrodi DV,Peever TL,Mavrodi OV,et al.Diversity and evolution of the phenazine biosynthesis pathway[J].Applied and Environmental Microbiology,2010,76(3):866-879
[10]Cui CY,Yang CX,Song SH,et al.A novel two-component system modulates quorum sensing and pathogenicity in Burkholderia cenocepacia[J].Molecular Microbiology,2018,108(1):32-44
[11]Tseng HC,Chen CW.A cloned ompR-like gene of Streptomyces lividans 66 suppresses defective melC1,a putative copper-transfer gene[J].Molecular Microbiology,1991,5(5):1187-1196
[12]Rodríguez H,Rico S,Díaz M,et al.Two-component systems in Streptomyces:key regulators of antibiotic complex pathways[J].Microbial Cell Factories,2013,12:127
[13]Chang HM,Chen MY,Shieh YT,et al.The cutRS signal transduction system of Streptomyces lividans represses the biosynthesis of the polyketide antibiotic actinorhodin[J].Molecular Microbiology,1996,21(5):1075-1085
[14]Macneil DJ,Gewain KM,Ruby CL,et al.Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector[J].Gene,1992,111(1):61-68
[15]Bierman M,Logan R,O’Brien K,et al.Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.[J].Gene,1992,116(1):43-49
[16]Wang W,Wang HS,Hu HB,et al.Overexpression of afsR and optimization of metal chloride to improve lomofungin production in Streptomyces lomondensis S015[J].Journal of Microbiology and Biotechnology,2015,25(5):672-680
[17]Sheng CL,Wang W,Hu HB,et al.Function of a methyltransferase gene lomo3 involved in the biosynthesis of lomofungin[J].Microbiology China,2016,43(3):575-582(in Chinese)盛超兰,王威,胡洪波,等.一个甲基转移酶基因lomo3在洛蒙真菌素生物合成途径中的功能[J].微生物学通报,2016,43(3):575-582
[18]Li S,Ye QM,Wang W,et al.High-performance liquid chromatography analysis of lomofungin in Streptomyces lomondensis[J].Journal of Liquid Chromatography&Related Technologies,2013,36(15):2059-2068
[19]Xie K,Peng HS,Hu HB,et al.OxyR,an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP72[J].Research in Microbiology,2013,168(10):646-653